The initial skin blood flow response to rapid local heating is an axon reflex, which may be mediated by calcitonin gene-related peptide and substance P released from C-fibres. We investigated the role of nitric oxide (NO) and noradrenaline on the temperature threshold for the axon reflex during gradual local heating. 36 subjects participated in two studies. Using microdialysis, we examined the following interventions: NO synthase inhibition (10 mMNG-nitro-L-arginine methyl ester, L-NAME); low-dose NO infusion (1.0 μM sodium nitroprusside, SNP); adrenergic blockade (10 mM bretylium tosylate); and low-dose (0.1 μM) noradrenaline infusion. Laser-Doppler flowmetry was used to measure red blood cell flux. Skin was heated at a rate of 0.1°C min−1 from 33°C to 40°C. Compared to control skin sites, the axon reflex response was shifted to a higher temperature in 4 subjects in the L-NAME sites (control, 37.0 ± 0.3°C, n= 16; L-NAME, 39.8 ± 0.1°C, n= 4; P < 0.001) and absent in 12 subjects. The response was also absent in L-NAME plus low-dose SNP sites and not altered by low-dose SNP infusion alone. Adrenergic blockade, with and without low-dose noradrenaline infusion, also abolished the axon reflex response in all subjects. Low-dose noradrenaline infusion alone shifted the axon reflex to a significantly lower temperature threshold compared to control sites (control, 38.2 ± 0.5°C; noradrenaline, 37.7 ± 0.4°C, P < 0.05, n= 5). These results suggest that endogenous NO and noradrenaline contribute to the temperature threshold of the axon reflex response during gradual local heating of the skin.